Recombinant transcription factors comprising the DNA binding domains from zinc finger proteins (“ZFPs”) or TAL-effector domains (“TALEs”) and engineered nucleases including zinc finger nucleases (“ZFNs”), TALENs, CRISPR/Cas nuclease systems, and homing endonucleases that are all designed to specifically bind to target DNA sites have the ability to regulate gene expression of endogenous genes and are useful in genome engineering and gene therapy. See, e.g., U.S. Pat. Nos. 9,045,763; 9,005,973; 8,956,828; 8,945,868; 8,586,526; 8,329,986; 8,399,218; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 20100218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et at (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy.
Nuclease-mediated gene therapy can be used to genetically engineer a cell to have one or more inactivated genes and/or to cause that cell to express a product not previously being produced in that cell (e.g., via transgene insertion and/or via correction of an endogenous sequence). Examples of uses of transgene insertion include the insertion of one or more genes encoding one or more novel therapeutic proteins, insertion of a coding sequence encoding a protein that is lacking in the cell or in the individual, insertion of a wild-type gene in a cell containing a mutated gene sequence, and/or insertion of a sequence that encodes a structural nucleic acid such as shRNA or siRNA. Examples of useful applications of ‘correction’ of an endogenous gene sequence include alterations of disease-associated gene mutations, alterations in sequences encoding splice sites, alterations in regulatory sequences and targeted alterations of sequences encoding structural characteristics of a protein. Transgene construct(s) is(are) inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, e.g., U.S. Pat. Nos. 9,045,763; 9,005,973; 7,888,121 and 8,703,489.
Clinical trials using these engineered transcription factors and nucleases have shown that these molecules are capable of treating various conditions, including cancers, HIV and/or blood disorders (such as hemoglobinopathies and/or hemophilias). See, e.g., Yu et al. (2006) FASEB J. 20:479-481; Tebas et at (2014) New Eng J Med 370(10):901. Thus, these approaches can be used for the treatment of diseases.
Severe combined immunodeficiency (SCID) is a heterogeneous group of primary immunodeficiencies comprising at least 11 different conditions (Kutukeuler et al (2012) It J of Ped 38:8). All patients with SCID are susceptible to infections from common bacteria and viruses as well as opportunistic and fungal pathogens. X-linked severe combined immunodeficiency (X-SCID) is an immunodeficiency disorder in which the body produces very few T cells and natural killer cells. In the absence of T cell help, B cells become defective (Fisher et al. (2002) Nature Reviews 2:615-621). It is an X-linked recessive trait such that nearly all patients are male, and stems from a mutated version of the IL2RG gene (also referred to as the “common gamma” gene or “common cytokine receptor gamma chain”), located at xq13.1 on the X-chromosome. The common gamma protein is shared between receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, leaving X-SCID patients unable to develop functional T and NK cells. Persons afflicted with X-SCID often have infections very early in life, before three months of age. This occurs due to the decreased amount of immunoglobulin G (IgG) levels in the infant during the three-month stage. This is followed by viral infections such as pneumonitis, an inflammation of the lung which produces common symptoms such as cough, fever, chills, and shortness of breath. Recurrent eczema-like rashes are also a common symptom. Other common infections experienced by individuals with X-SCID include diarrhea, sepsis, and otitis media. Some other common symptoms that are experienced by X-SCID patients include failure to thrive, gut problems, skin problems, and muscle hypotonia (Vickers Severe combined Immune Deficiency: Early Hospitalisation and Isolation (2009) pp. 29-47. ISBN 978-0-470-31986-4). Without therapeutic and/or environmental intervention, X-SCID is typically fatal during the first year of life (Hacein-Bey-Abina et al, (2002) NEJM 346: 1185-1193).
Another type of SCID is related to defects in the recombination activating genes (RAG1, RAG2), where approximately 10% of all SCID cases are tied to RAG1 or RAG2 (Ketukculer, ibid). The protein products of the RAG1 and RAG2 genes (Rag1 and Rag2, respectively) are essential for V(D)J rearrangement in B and T cell receptors, and thus are required for proper development of B cells and T cells and are also thought to be involved in inflammation (see, e.g., U.S. Patent Publication No. 20110016543). Together, Rag1 and Rag2 initiate V(D)J recombination by cleaving DNA to generate double strand breaks which are then repaired by the NHEJ machinery.
Omenn Syndrome is an autosomal recessive variant of SCID with distinctive clinical features of generalized erythodermia, hepatosplenomegaly and lymphadenopathy. Unlike patients with classic SCID, patients with Omenn Syndrome have circulating T cells with an abnormal phenotype: they are typically poorly reactive, oligoclonal, and display cell-surface markers of previous activation. B cells are typically absent or low and IgG levels are generally low while IgE levels are high (Matthews et al (2015) PLoS One 10(4):e0121489). Omenn Syndrome is typically caused by mutations in RAG1 or RAG2 although mutations in other genes can also lead to it. Generally, hypomorphic RAG mutations, sometimes in combination with RAG null mutations, lead to Omenn Syndrome (Matthews, ibid).
Currently, there are three types of treatments available for SCID patients, namely, the use of medication, sterile environments, and intravenous immunoglobulin therapy (IVIG). First, antibiotics or antivirals are administered to control opportunistic infections, such as fluconazole for candidiasis, and acyclovir to prevent herpes virus infection (Freeman et al. Current Opinion in Allergy and Clinical Immunology (2009) 9 (6):525-530). In addition, the patient can also undergo intravenous immunoglobulin (IVIG) supplementation. However, the IVIG is expensive, in terms of both time and money. In addition, the aforementioned treatments only serve to prevent opportunistic infections, and are by no means a cure for X-SCID or other SCID disorders.
At present, bone marrow transplantation (BMT) is the standard curative procedure and results in a full immune reconstitution, if an appropriate donor can be identified and if the engraftment is successful. A bone marrow transplant requires an acceptable human leukocyte antigen (HLA) match between the donor and the recipient. As the array of HLA molecules is different between individuals, cells of the immune system can utilize the HLA apparatus to distinguish self from foreign cells. A BMT can be allogeneic (donor and recipient are different people) or autologous (donor and recipient are the same person). An autologous BMT therefore has a full HLA match, whereas, a match for an allogenic BMT is more complicated. In standard practice, an allogenic graft is better when all 6 of the known major HLA antigens are the same—a 6 out of 6 match. Patients with a 6/6 match have a lower chance of graft-versus-host disease, graft rejection, having a weak immune system, and getting serious infections. For bone marrow and peripheral blood stem cell transplants, sometimes a donor with a single mismatched antigen is used—a 5 out of 6 match. Therefore, a BMT may result in a full immune reconstitution and thus be curative in an X-SCID patient, but potential complications limit efficacy and widespread use. For patients with Omenn Syndrome, BMT is also the preferred method of treatment however Omenn Syndrome patients have a higher rate of mortality following BMT than other SCID patients.
Previous gene therapy clinical trials for X-SCID patients have used retroviral vectors comprising a wild type IL2RG gene (Cavazzana-Calvo et al. (2000) Science 288(5466):669-72). Retroviral vectors randomly integrate into the host genome, however, and thus can cause insertional oncogenesis in patients when integration occurs in proto-oncogenes (Hacein-Bey-Abina et al. (2008) J. Clin Investigation 118(9):3132-42). The majority of patients undergoing this therapy developed leukemia as a result of this insertional oncogenesis, thus this method is not a safe and effective therapy.
The development of integrase-deficient lentiviral vectors (IDLV) (Philippe et al. (2006) Proc. Nat'l Acad. Sci. 103(47):17684-9), or IDLV, has facilitated further investigation of gene correction of IL2RG for X-SCID due to its inability to integrate into the host genome. The ability of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas systems and TtAgo to target a specific region of DNA, introduce a targeted double stranded break, which then facilitates targeted integration of an introduced transgene makes this genome editing technology highly attractive in the development of a potential curative treatment.
To this end, investigators have targeted exon 5 of the endogenous IL2RG locus for ZFN cleavage and subsequent TI of IDLV-delivered corrective IL2RG cDNA in hematopoietic stem and progenitor cells (HSPCs). See, e.g., U.S. Pat. Nos. 7,888,121 and 7,951,925; Lombardo et al. (2007) Nat Biotech 25(11):1298-306; Genovese et al. (2014) Nature 510(7504):235-40. However, these methods may have potential disadvantages in that introducing a transgene in the middle of an exon creates a partially transcribed region upstream of the introduced transgene, which may interfere with the activity of the introduced corrective gene. Furthermore, the delivered episomal transgene may still be able to randomly integrate into the genome if another viral integrase is present in the cell. Immunosuppressed patients (such as all X-SCID patients are) might have activation of endogenous retroviruses, thus barring patients who are also HIV positive from receiving virally delivered gene therapy for X-SCID treatment.
Thus, there remains a need for additional strategies of IL2RG and RAG gene correction and transgene donor delivery for treatment and/or prevention of SCID.